4 Journal of Comparative Neurology, 



blue could be seen to pass from the metamere injected through several 

 adjacent metameres. When the body wall began to be distended, an in- 

 jection was made in another region and this was repeated until the en- 

 tire body cavity seemed to contain the blue. At iirst the attempt was 

 made to force in as much fluid as possible at one injection, but this 

 injured the tissues so that it seriously interfered with the circulation. 

 Then injecting a smaller amount and repeating the injection after a 

 short interval was tried— a method also used by Meyer ('96); this al- 

 ways gave very satisfactory results. Usually each worm was injected 

 three times at intervals of about 40 min., using each time great care 

 not to injure the animal more than was absolutely necessary. After 

 the first injection, much more of the stain could be forced in at a time 

 without danger of rupturing the body wall. 



Following a suggestion given me by Dr. G. C. Huber, the animals 

 were put away in the dark after each injection. It was found that, if 

 two worms of equal size and apparent vigor were injected with equal 

 amounts of the same methylene blue solution, the one kept in the dark 

 gave surprisingly richer results than the one kept in the light. Instead 

 of leaving the injected worm directly exposed to the air, it was found 

 better to keep it in its normal medium— the sea-water. About four 

 hours after the first injection — a shorter time on very warm days— the 

 worms were taken out and, without being opened, exposed to the hght 

 and air. At first the color of the body would be nearly normal, but 

 in about fifteen minutes it would become a rich blue. I am, however, 

 inclined to believe that this exposure causes merely a bluing of the 

 general tissues and that the nerve elements are already stained when 

 the animal is removed from the dark and the sea-water. Several times 

 a worm was removed from the dark and while it was still in the sea- 

 water, a parapodial cirrus was cut off. No matter how quickly this was 

 placed under the microscope, the nerve-cells of the sense-organs were 

 already blue. Sometimes a worm would be so laid that only the para- 

 podia of one side would be directly exposed to the air, yet the nerve tis- 

 sues of the parapodia from the opposite side would be as richly stained 

 as those from the exposed side. Then, again, several times the brain 

 was richly stained in worms that had not been, opened in this region. 

 These facts seem to indicate that direct exposure of the nerve tissues 

 themselves to the air is not always necessary. 



My own observations have led me to the conclusion that the main 

 factors in obtaining a rich stain in Nereis are: i. vigorous, healthy 

 worms ; 2. the injection of a large quantity of strong stain in such a 

 manner as to avoid interfering with the circulation ; and 3. the keeping 



